Viral and Microbial RNA Modifying Enzymes

病毒和微生物 RNA 修饰酶

• 批准号: 8449021
• 负责人: Stewart H Shuman
• 金额: $ 52.01万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8449021
• 关键词: ATP-Binding Cassette Transporters; Active Sites; Amoeba genus; Anti-Infective Agents; Antiviral Agents; Bacteria; Bacterial RNA; Bacteriophage T4; Bacteriophages; Binding; Binding Proteins; Biochemical Reaction; Biological Models; C-terminal; Catalytic Domain; Cells; Cellular Stress; Clostridium thermocellum; Coupled; DNA Viruses; Diphosphates; Enzymes; Escherichia coli; Event; Evolution; Family; Funding; Genetic; Genetic Models; Genetic Translation; Genomics; Glutamic Acid-Specific tRNA; Grant; Guanine; Healed; Homologous Gene; Human; Hydrolysis; Immune response; Infection; Ligase; Light; Lysine-Specific tRNA; Mammalian Cell; Manganese; Maps; Mediating; Messenger RNA; Methyltransferase; Modification; N-terminal; Open Reading Frames; Operon; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Phylogenetic Analysis; Phylogeny; Polynucleotide 5' -Hydroxyl-Kinase; Poxviridae; Property; Proteins; Protozoa; RNA; RNA Caps; RNA Ligase (ATP); RNA Processing; RNA triphosphatase; Reaction; Research; Ribose; Saccharomycetales; Smallpox; Specificity; Structure; Substrate Specificity; System; Taxon; Toxin; Transfer RNA; Vaccinia; Vaccinia virus; Vaccinium; Viral; Viral Proteins; Virus; Virus Diseases; Work; Yeasts; anticodon nuclease; cytotoxicity; drug discovery; fungus; guanylyltransferase; healing; in vivo; insight; killings; mRNA Precursor; mRNA Stability; mRNA capping; mRNA guanylyltransferase; methyl group; microbial; novel; nuclease; nucleoside triphosphatase; pathogen; plant fungi; polypeptide; programs; public health priorities; repair enzyme; repaired; response; restriction enzyme; seal; tripolyphosphate; weapons

DESCRIPTION (provided by applicant): PROJECT SUMMARY: This project aims to illuminate the mechanisms and structures of viral enzymes that cap the 5' end of messenger RNA. The m7GpppN cap is a distinctive feature of eukaryal cellular and viral mRNA that is required for mRNA stability and translation. Capping entails three enzymatic reactions: (i) the 5' triphosphate of the pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase (TPase); (ii) the diphosphate RNA end is capped with GMP by RNA guanylyltransferase (GTase); and (iii) the GpppN cap is methylated by RNA (guanine-N7) methyltransferase (MTase). Whereas the three capping reactions are universal in eukarya and DNA viruses, there is a remarkable diversity in the genetic organization of the cap- forming enzymes in different taxa and in different viruses, as well as a complete divergence in the structure and catalytic mechanism of the TPase enzymes found in fungi, protozoa, and several large DNA viruses versus the human TPase. These differences can be exploited to develop novel anti-infective agents directed against capping of the pathogen's mRNAs. They also provide instructive clues to eukaryal phylogeny and evolution of large DNA viruses. This proposal focuses on the poxvirus mRNA capping enzyme, a heterodimer composed of TPase, GTase and MTase domains fused within a single large polypeptide subunit, plus a smaller subunit that binds and stimulates the MTase domain. We plan to functionally map the active sites and domain interfaces of the poxvirus capping enzyme, guided by a new crystal structure of the complete capping enzyme heterodimer. As an outgrowth of our studies of RNA end-modification by viral enzymes, we've developed a new line of research into end-healing and end-sealing enzymes that repair programmed RNA breaks. Programmed RNA damage is an ancient mechanism of responding to cellular stress and distinguishing self from non-self. RNA damage also figures prominently in host responses to virus infection. tRNA damage inflicted by a latent anticodon nuclease PrrC (which breaks tRNALys) underlies a potent host innate immune response to bacteriophage T4 infection, which is thwarted by a virus-encoded tRNA repair system consisting of T4 Pnkp (polynucleotide kinase/phosphatase) and T4 Rnl1 (RNA ligase 1). We have dissected the mechanism, structure, and specificity of Pnkp and Rnl1, and we have extended our analysis to discover or characterize new RNA repair enzymes from viruses, bacteria and human cells. RNA damage and repair now comprise a significant component of the research effort supported by this grant. The current plan focuses on two aspects of the host-pathogen RNA damage/repair dynamic: (i) genetic and structural analysis of PrrC anticodon nucleases and (ii) characterization of a newly discovered two-component RNA repair system (Hen1-Pnkp) that is distributed widely among bacteria.

描述（由申请人提供）： 项目摘要：该项目旨在阐明限制信使RNA 5'端的病毒酶的机制和结构。 M7GPPPN帽是真核细胞和病毒mRNA的独特特征，是mRNA稳定性和翻译所需的。限额需要三个酶促反应：（i）通过RNA三磷酸酶（TPase）将前MRNA的5'三磷酸化为双磷酸盐； （ii）双磷酸RNA端由RNA Guanylyllansferase（GTase）用GMP限制； （iii）GPPPN帽被RNA（鸟嘌呤-N7）甲基转移酶（MTase）甲基化。尽管这三个上限反应在真核病和DNA病毒中是普遍的，但在不同分类单元和不同病毒中的盖酶的遗传组织中存在显着的多样性，以及在结构和催化机制中的完全差异与人TPase相比，在真菌，原生动物和几种大型DNA病毒中发现的TPase酶。可以利用这些差异来开发针对病原体mRNA封盖的新型抗感染剂。他们还为大型DNA病毒的真核系统发育和演变提供了有益的线索。该提案的重点是痘病毒mRNA限制酶，该酶是由TPase，GTase和MTase结构域组成的异二聚体，该二聚体融合在单个大多肽亚基中，再加上结合和刺激MTase域的较小亚基。我们计划在功能上绘制痘病毒封盖酶的活动位点和域界面，并由完整封盖酶异二聚体的新晶体结构进行指导。 作为我们对病毒酶进行RNA终端修饰的研究的产物，我们已经开发了一系列新的研究，用于修复编程RNA断裂的最终治疗和最终密封酶。编程的RNA损伤是对细胞应力反应并区分自我的古老机制。 RNA损伤在宿主对病毒感染的反应中也显着。 tRNA损伤是由潜在的反ododon核酸酶PRRC（打破TRNALYS）是对噬菌体T4感染的有效宿主先天免疫反应，该反应由病毒编码的tRNA修复系统挫败，该系统由T4 PNKP（由T4 PNKP）（多核苷酸激酶/磷酸酶）和T4 RNL1组成（RNA连接酶1）。我们已经阐述了PNKP和RNL1的机制，结构和特异性，并扩展了分析，以发现或表征来自病毒，细菌和人类细胞的新RNA修复酶。现在，RNA损坏和维修构成了该赠款支持的研究工作的重要组成部分。当前的计划侧重于宿主 - 病原RNA损伤/修复动态的两个方面：（i）PRRC反密码子核酸酶的遗传和结构分析，以及（ii）新发现的两组分组RNA修复系统（HEN1-PNKP）的表征被广泛分布在细菌中。